Structure and function of proteins: Pseudo-contact shifts as a valuable tool in NMR spectroscopy
Researchers of the Biozentrum and the Department of Chemistry have developed a novel NMR method with which they could track the dynamics of the activation of a G-protein-coupled receptor at the molecular level for the first time. These physiologically important membrane proteins are important molecular switches that play a central role in mediating the signaling cascade from the outside to the inside of the cell and are therefore targeted by numerous drugs. The findings were recently published in “Science” and may provide knowledge for the rational design of new drugs and are an impressive example of the potential of the new NMR method for future applications.
Please note: A separate news article has been published on the above-mentioned Science article. This article focuses more on the underlying NMR method.
NMR spectroscopy is the method of choice for determining the structure, dynamics and interaction of biomacromolecules such as proteins in solution. Valuable information about the different conformations of biomacromolecules can be derived from the changes in the so-called chemical shift (the position of the signals in the NMR spectrum). In the work published in Science, the function of G-protein-coupled receptors (GPCRs) can be understood by precisely determining the population of the individual conformations in the coupled equilibria between inactive, preactive and active states. In contrast, static structures, such as those determined by crystallographic studies, do not provide comparable information on function.
Lanthanide tag induces chemical shift change
The change in the chemical shift can be caused by pseudo-contact shifts which are induced by the incorporation of “paramagnetic tags” into the protein. The tags are cage molecules that contain a paramagnetic metal ion from the lanthanide series, such as dysprosium, terbium or thulium, which causes the signal shifts by interacting with the NMR-active atomic nuclei of the protein (e.g. 1H, 13C, 15N). The tag is site-specifically incorporated through a chemical reaction of the tag with certain amino acid residues of the protein.
Classic methods of protein NMR spectroscopy reach their limits when applied to larger proteins (>100 kDa). In addition, the method using pseudo-contact shifts is not suitable for all proteins as, for example, several suitable reaction sites for the incorporation of the tag may be present. In other cases, the protein activity and/or its structure are too much disturbed by the introduction of the tag.
Tagged binding partner for indirect protein analysis
Research in the group led by Prof. Daniel Häussinger at the Department of Chemistry is focused on the synthesis and application of latest-generation lanthanide tags. In cooperation with the research group of Prof. Stephan Grzesiek und Dr. Feng-Jie Wu from the Biozentrum, Dr. Pascal Rieder succeeded in applying the new NMR method on GPCRs by not tagging the protein directly, but a so-called nanobody (the smallest functional fragment of an antibody) that binds to the protein. When the tagged nanobody binds to the protein, pseudocontact shifts are induced and the protein can thus be measured indirectly. Nanobodies are known for many proteins, which could allow for a variety of future applications of this method in protein analysis. The researchers published the method in the “Journal of the American Chemical Society” and reported on the structural analysis of a GPRC, in which they obtained NMR data with a significantly higher information density than from previous NMR measurements. In a second publication recently published in “Science”, they were also able to use the method to dynamically map the functioning of a GPCR for the first time and thus determine and understand different activity states of the protein.
GPCRs act as molecular switches that control important physiological processes by transmitting a signaling cascade into the interior of cells and are therefore targeted by numerous drugs. Because of their size and the dynamic equilibrium between different states, GPCRs have so far been difficult to access by NMR spectroscopy. Therefore, the work presented closes the gap between the known static structures of these proteins and their function.
Original publications
Feng-Jie Wu, Pascal S. Rieder, Layara Akemi Abiko, Philip Rößler, Alvar D. Gossert, Daniel Häussinger, Stephan Grzesiek
Nanobody GPS by PCS: An Efficient New NMR Analysis Method for G Protein Coupled Receptors and Other Large Proteins
Journal of the American Chemical Society 2022, 144 (47), 21728-21740; doi: 10.1021/jacs.2c09692
Feng-Jie Wu, Pascal S. Rieder, Layara Akemi Abiko, Anne Grahl, Daniel Häussinger, Stephan Grzesiek.
Activation dynamics traced through a G protein coupled receptor by 81 1H-15N NMR probes
Science (2025), doi: 10.1126/science.adq9106
Further Information
Website research group Prof. Daniel Häussinger
Website research group Prof. Stephan Grzesiek
Website research group Dr. Feng-Jie Wu